Both overexpression and knock-out deletion of granulocyte-macrophage colony stimulating factor (GM-CSF) in mice can lead to dysregulation of myeloid differentiation and autoimmune disease. This suggests that GM-CSF signaling in myeloid cells is a critical temporally and/or quantitatively regulated mechanism for preventing immunopathogenesis. Like many cytokines involved in hematopoiesis, GM-CSF uses the JAK2-STAT5 signal transduction/transcriptional regulatory pathway to exert its control over cellular functions. Naturally occurring STAT5 dominant negative and activator isoforms make this pathway a key 'switch' mechanism for both myeloid differentiation stage-specific and monocyte/macrophage activation-specific responses to GM-CSF. GM-CSF activation of truncated STATS suppresses precocious activation ofgene expression in immature myeloid cells. In GM-CSF direct stimulation of mature macrophages and in endotoxin activation ofmonocytes, GM-CSF activates full-length STAT5 isoforms that upregulate gene transcription. We have found that high autocrine production of GM-CSF by at-risk/Type 1 diabetic human monocytes and nonobese diabetic (NOD) mouse macrophages promotes a build up of tyrosine phosphorylated STAT5. Moreover, the activated STATS isoforms in these autoimmune cells have stage-specific and isoform-specific alterations in their DNA binding capabilities: truncated repressor isoforms are unable to bind DNA in unactivated antoimmune monocytes, and prolonged binding of full-length activator isoforms in autoimmune macrophages. Our preliminary data show that congenic strain allele replacement of a Chromosome 11 lcM locus around the GM-CSF gene, and not the region containing the STAT5A & B genes, can reverse prolonged STAT5 tyrosine phosphorylation in NOD monocytes and macrophages as well as the diminished DNA binding capacity of STAT5 truncated isoforms in NOD monocytes. This region includes the Idd4.2 susceptibility locus and several known genes for cytokines whose effects can be mediated through their activation of STATS. However after activated by GM-CSF, persistent STATS phosphorylation and DNA binding in NOD macrophages become independent of GM-CSF and Jak2/3 kinase activity. This suggests that other STATS regulatory mechanisms are dysfunctional in NOD myeloid cells, preventing STATS deactivation and possibly hindering the cell's responsiveness to subsequent signaling by other cytokines important in myeloid differentiation and activation, including macrophage-colony stimulating factor (M-CSF) and granulocyte-colony stimulating factor (G-CSF), and IL-10. This proposal outlines our plan to use NOD & NOD congenic recombinant strain mice 1) to determine the importance of GM-CSF differential activation of defective STAT5 isoforms in the subsequent responsiveness of NOD myeloid cells to other cytokines, 2) to phenotypically define & narrow the GM-CSF/Idd4.2-containing Chll interval and substantiate its importance in NOD STAT5 dysfunction, and 3) to investigate the underlying cause(s) promoting STAT5 GM-CSF/Jak independence and preventing STAT5 deactivation after GM-CSF stimulation of NOD monocytes and macrophages.